Rabies vaccine preparation

ABSTRACT

A rabies vaccine is prepared by adaptation of the rabies virus to chick-embryo fibroplast cell cultures in progressing dilutions of from 10 -1  to 10 -5  for the inoculation of the cell culture passages and for multiplication in dilutions of from 10 -2  to 10 -5  for inoculation of the growth cultures.

This invention relates to a rabies vaccine and to a process for itspreparation.

Inactivated vaccines and virus vaccines for the immunization againstrabies are known. The inactivated rabies vaccines consist of suspensionsof virus-containing central nervous tissue of infected animals, such asrabbits or sheep, or of suspensions of infected duck fetuses. Vaccinesof this type have the draw-back that, due to the high content of foreignproteins, they may cause undesired side effects at the point ofinjection as well as of general nature. When rabies vaccines from thecentral nervous tissue are used, the patient may even suffer fromneurocomplications with permanent damage, all the more since a series ofinjections is required for a sufficient protection against rabies. Ithas been ascertained that similar side effects also occur with animals.

Attempts have been made to propagate the rabies virus in tissue culturesof cell strains of baby hamster kidneys, for example BHK 21-cells, inNIL cells, in primary cells of hamster kidneys, or in chick-embryofibroblast cells, and to prepare therefrom inactivated vaccines foranimals. To obtain rabies virus vaccines for animals, primary cells ofkidneys of pigs or hamsters or cell strains of kidney cells of dogs orbovine are being used. In these cases, the virus concentrations asobtained with central nervous system material cannot be reached. Asufficiently high virus titer is, however, the prerequisite to aneffective rabies vaccine, especially to an inactivated vaccine. Some ofthe cells used have the further drawback that they may cause malignanttumors.

It is not possible to use a serum yielding higher concentrations ofvirus during the growth of the virus in the cell culture, as the foreignserum would lead to the formation of undesired antibodies. To overcomethis difficulty, an additional procedure of concentrating thevirus-containing solution, for example a precipitation, centrifugationor ultra-filtration, has been inserted into the production line, i.e.measures by which the process of preparation becomes more difficult andmore expensive.

In the case of test vaccines for use in animals, maximum titers of 10⁵.0to ⁶.0 PFU/ml (plaque forming units/ml) or of 6.1 × 10⁶ baby mouse LD₅₀/ml (LD = lethal dose) have been described with chick-embryo fibroblastcells, i.e. titers which do not ensure a good immunization.

It is an object of the present invention to provide a process for thepreparation of a rabies vaccine by adapting the rabies virus to primarychick-embryo fibroblast cells and propagating same in primarychick-embryo fibroblast cells after adaptation, which comprises usingthe rabies virus for adaptation in increasing dilutions of from 10⁻¹ to10⁻⁵ and for inoculation of the multiplication cultures in dilution inthe range of from 10⁻² to 10⁻⁵.

According to a preferred embodiment the process is carried out at atemperature in the range of from 30° to 38° C, more preferably 32° to37° C.

For the process of the invention there can be used all rabies strainsgenerally used in the preparation of rabies virus vaccines, for examplethe Flury, LEP and Flury HEP strains, the rabies virus fixe strain VP11, the fixe strain Pasteur and the PM strain (PM = Pitman-Moore).

Chick-embryos for the fibroblast cells are cheap, readily acessible andthe yield of cells is very good. For use in man there are preferablyused chick-embryos of so-called SPF eggs, i.e. eggs of chickensspecifically raised free from pathogens in chicken farms run for thispurpose. Tissue cultures of SPF eggs are free from undesired foreignviruses as can be observed in eggs of chickens bred in usual manner forexample the virus of infectious bursitis, of infectiouslaryngotracheitis, of influenza type A, of chicken pox, aviaryencephalomyelitis, of inclusion hepatitis, of infectious bronchitis,chicken leucosis, of the Marek disease, of the Newcastle disease, ofaviary reoviruses, of aviary adeno-viruses, of Mycoplasma gallisepticum,of Mycoplasma synoviae and of Salmonella pullorum. The use ofchick-embryo fibroblast cells for the preparation of a rabies vaccinefor man is novel and not yet described.

The rabies vaccine is prepared in accordance with the invention byadapting the rabies virus to primary chick-embryo fibroplast cells,propagating the adapted virus in primary chick-embryo fibroplast cellsand working up the virus-containing suspension, optionally afterinactivation in known manner to obtain a vaccine. The rabies virusadapted according to the invention allows of obtaining in the viruspropagation such high virus concentrations that a useful rabies vaccinecan be prepared therefrom.

As starting material mammal brain is preferably used, especially thebrain of rabbits, containing, for example, viruses of the strain VP 11,the strain Pasteur, or the strain PM, or homogenized chick-embryomaterial containing for example viruses of the strain Flury LEP or FluryMEP, which is applied to a monolayer cell culture of primarychick-embryo fibroblast cells.

When an undiluted virus material is transferred according to state ofthe art to infect as high a number of cells as possible, a few cells arepopulated only and in the following passages the number of infectedcells does not increase; it is even reduced. Hence, an adapting of thevirus to the cell does not take place and cannot be obtained in thismanner.

As compared therewith, when the virus-containing suspension to be usedfor the passages is diluted to 10⁻¹ to 10⁻⁴, a rabies virus is obtainedwhich is suitable for the preparation of a rabies vaccine. The decisivefact for the transfer of one dilution to the next higher one is themultiplication kinetics of the virus in the preceding passage. In viewof the fact that virus strains can be well propagated at a dilution of10⁻², there are first made passages with the 10⁻¹ and 10⁻² dilutions ofthe starting material.

To control the virus multiplication, infested cover-glass cultures inLeighton tubes, which have been treated in similar manner, are used.When the cover-glass culture is colored with a fluorescin-markedanti-rabies gamma globulin, the dyed preparation can be evaluated underthe fluorescence microscope, the percentage of the cell infested withrabies virus indicating the muliplication kinetics. A sufficient numberof controls is prepared so that the virus population of the cells can befollowed daily. For each reading 1 to 2 tubes are required.

At first, the viruses settle very relunctantly on the cells but duringthe course of the passages according to the invention the settlementincreases.

When, in one passage, 100% of the cells are infested with virus within 3to 4 days, the 10⁻² or 10⁻³ dilution is used and, thereafter, the 10⁻⁴dilution, if necessary. With the change to higher dilutions, the virustiter increases during the passages with a settlement of 100% of thecell monolayer.

The virus-containing suspension of the last passage is used as seedmaterial for the virus growth in the preparation of the vaccine. Owingto the high titer of the seed material and the rapid settlement of thecells by the adaptation according to the invention, very little seedmaterial is required in the production so that the valuable material islargely saved. The extensive tests requested by the governments highlyincrease the costs of the seed material.

To obtain a sufficiently high virus titer in the manufacture of thevaccines 1 LD₅₀ of the seed material is required for about 20,000 cells,whereas about 1 to 10 LD₅₀ or PFU (plaque forming unit) are necessaryaccording to the state of the art to inoculate one cell.

LD₅₀ in the mouse and PFU in tissue are approximately comparable.

The adaptation is reliable and rapid. The virus titers obtained are byten times or more higher than the titers obtained according to the stateof the art. Hence, concentration procedures can be dispensed with. If,however, a concentration step is added, for example for vaccines forman, the virus titer can again be increased to the hundred-fold.

The following examples illustrate the invention.

EXAMPLE 1

Adaptation of a rabies virus and preparation of an inactivated vaccinefor use in man and animals.

As starting material the rabies virus fixe strain VP 11 was used, whichcan be obtained from the World Health Organization, Reference Center forRabies, Institute Pasteur, Paris, in the form of a lyophilized substancefrom rabbit brain.

The virus had a titer of 10⁵.2 LD₅₀ /ml, measured by intracerebralinjection into white mice of 11 to 15 grams of the family NMRI.

The content of the ampoule was dissolved in 1 ml of aqua destillata andapplied on primary chick-embryo fibroblast cells (CEFC) in a dilution of10⁻².

Seven cover-glass cultures of the same cell preparation in Leightontubes were also inoculated with the 10⁻².0 dilution (passage I 1). Aftera time of incubation of 5 days at +37° C, less than 25% of thefibroblast cells had been infected.

The virus-containing cell-free supernatant in the 10⁻¹.0 dilution wastransferred to the 2nd passage of primary chick-embryo fibroblast cells(passage I 2). After a time of incubation of 6 days at +37° C, about 25%of the cells were infested. The cell-free supernatant was transferred inthe dilutions 10⁻¹.0 and 10⁻².0 onto primary CEFC (passage I 3). After atime of incubation of 4 days, with the 10⁻¹.0 dilution 70% and with the10⁻².0 dilution 90% of the cells were infested. The passage with the10⁻¹.0 dilution was continued with this dilution and the passage carriedout with the 10⁻².0 dilution was continued in that dilution.

A new series of passage was branched off, the passage carried through inthe 10⁻².0 dilution, which was continued with the 10⁻³.0 dilution(passage I 4).

The further course of adaptation is illustrated by the following table:

    ______________________________________                                                                      virus                                           passage                                                                             dilu-  harvest/ % infested                                                                            titer                                           No.   tion   day      cells   LD.sub.50 /ml                                                                         remarks                                 ______________________________________                                        I 5   10.sup.-1                                                                            3        75                                                            10.sup.-2                                                                            3        100                                                           10.sup.-3                                                                            3        100                                                     I 6   10.sup.-1                                                                            3        75                                                            10.sup.-2                                                                            3        100                                                           10.sup.-3                                                                            3        75                                                      I 7   10.sup.-1                                                                            4        50                                                            10.sup.-2                                                                            4        75                                                            10.sup.-3                                                                            4        100                                                     I 11  10.sup.-1                                                                            5        25                                                            10.sup.-2                                                                            5        50                                                            10.sup.-3                                                                            5        50                                                                                           adaptation                               I 12  10.sup.-1                                                                            3        25             of virus                                       10.sup.-2                                                                            3        50             stain still                                    10.sup.-3                                                                            3        50             insuffi-                                                                      cient                                    I 13  10.sup.-2                                                                            3        25                                                            10.sup.-3                                                                            3        75                                                      I 17  10.sup.-2                                                                            3        80      10.sup.7.1                                                                           adaptation                                     10.sup.-3                                                                            3        100     10.sup.7.6                                                                           of virus                                                                      strain still                             I 18  10.sup.-2                                                                            3        75      10.sup.6.9                                                                           insuffi-                                       10.sup.-3                                                                            3        95      10.sup.7.2                                                                           cient                                    I 19  10.sup.-2                                                                            3        50      10.sup.6.1                                            10.sup.-3                                                                            3        95      10.sup.6.2                                      I 26  10.sup.-2                                                                            3        90      10.sup.7.1                                            10.sup.-3                                                                            3        100     10.sup.8.0                                      I 27  10.sup.-2                                                                            3        100     10.sup.6.3                                            10.sup.-3                                                                            3        100     10.sup.7.4                                      I 28  10.sup.-2                                                                            3        100     10.sup.7.2                                            10.sup.-3                                                                            3        100     10.sup.7.8                                      I 29  10.sup.-2                                                                            3        90      10.sup.6.8                                            10.sup.-3                                                                            3        100     10.sup.7.6                                      I 30  10.sup.-2                                                                            3        90      10.sup.6.9                                            10.sup.-3                                                                            3        100     10.sup.8.0                                      I 31  10.sup.-2                                                                            3        90      10.sup.6.4                                                                           strain suf-                                    10.sup.-3                                                                            3        100     10.sup.7.8                                                                           ficiently                                                                     adapted                                  I 31  10.sup.-3                                                                            3        100     10.sup.8.0                                                                           1. repetition                            I 31  10.sup.-3                                                                            3        100     10.sup.8.6                                                                           2. repetition                            ______________________________________                                    

Passage I 31 constitutes the seed material for an inactivated rabiesvirus ad.us.hum. It is surprising that in the passages I to 26 to I 31the virus concentration of the 10⁻³.0 dilutions is on the average about9 times higher than the concentration of the 10⁻².0 dilutions. This factis confirmed by repeating twice the passage I 31. In the firstrepetition the virus concentration was 40 times higher and in the second100 times higher.

Preparation of a vaccine ad us. hum.

Test vaccines were prepared from seed material of the strain Flury LEPadapted to primary chick-embryo fibroblast cells. The seed material,prepared according to the process specified above, was placed in a10⁻³.0 dilution on primary CEFC and incubated at +32° C. Afterclarification in a continuous flow centrifuge, the harvest of thevirus-containing suspension yielded a virus titer of 10⁷.4 LD₅₀ /ml.

The virus was then inactivated in known manner with β-propiolactone andthe vaccine was tested in the NIH test, a mouse-protection test, bycomparing it with a standard vaccine having an antigen value of 1.

The virus titer could be further increased. To this end, the virussuspension obtained was further purified in the continuous densitygradient and concentrated. In this manner a concentration factor ofabout 100:1 and a virus titer of 10⁹.3 LD₅₀ /ml were obtained. Theconcentrated virus suspension was adjusted to a concentration of 10:1,inactivated with β-propiolactone and likewise subjected to the NIH test.

The vaccines prepared in accordance with the invention had an antigenvalue of 1.2 and the vaccines with the 10:1 concentrated virus had anantigen value of 4.8.

It can be seen that the process of the invention makes it possible toprepare a vaccine having a sufficient antigen value for use in man. Inthe NIH test active vaccines are set free the antigen value of whichamounts to at least 30% of the antigen value of a standard vaccine. Thevaccine prepared as described above was 1.2 times better than thestandard vaccine A 18 tested under identical conditions, which isstandardized on the International World Health Organization rabiesstandard vaccine, and 4.8 times better with the purified andconcentrated virus material.

EXAMPLE 2

Adaptation of a production strain and preparation of a rabies virusvaccine for use in animals.

As starting material the rabies virus strain Flury HEP was used, whichcan be obtained from the American Type Culture Collection in Rockville,Md., USA, in the form of a homogenized and lyophilized substance fromvirus-containing chick-embryos. The virus was adapted in the mannerdescribed in Example 1, with the exception that the cultures wereincubated at 32° C and a dilution stage of 10⁻⁴.0 was additionally used.The adaptation of the virus strain HEP took place as follows:

    ______________________________________                                                                      virus                                           passage                                                                             dilu-  harvest/ % infested                                                                            titer                                           No.   tion   day      cells   LD.sub.50 /ml                                                                         remarks                                 ______________________________________                                        K  1  10.sup.-2                                                                            4        25      --                                                    10.sup.-3                                                                            3        74      10.sup.5.7                                      K  2  10.sup.-2                                                                            4        25      --                                                    10.sup.-3                                                                            4        70      10.sup.6.5                                      K  3  10.sup.-2                                                                            5        25      --     strain not yet                                 10.sup.-3                                                                            5        25      10.sup.4.8                                                                           adapted                                  K  7  10.sup.-2                                                                            4        100     --                                                    10.sup.-3                                                                            4        100     10.sup.7.5                                      K  8  10.sup.-3                                                                            4        100     10.sup.6.8                                                                           test vaccine                                   10.sup.-4                                                                            4        90      10.sup.7.3                                                                           prepared                                 K  9  10.sup.-3                                                                            4        100     10.sup.7.3                                                                           test vaccine                                   10.sup.-4                                                                            4        90      10.sup.7.5                                                                           prepared                                 K 12  10.sup.-3                                                                            3        100     10.sup.6.7                                            10.sup.-4                                                                            4        100     10.sup.7.3                                      K 13  10.sup.-3                                                                            3        100     10.sup.7.0                                            10.sup.-4                                                                            3        100     10.sup.7.5                                      ______________________________________                                    

It can be seen that in this case, too, higher virus titers can beobtained when the next higher dilution of the inoculation material isused for the following passage. Two test vaccines were prepared asfollows:

33% of a stabilizer solution consisting of polymerized and decomposedgelatin, which had been prepared as described in German Pat. No.1,118,792, and sodium glutamate were added to the virus-containingmedium of passages K8 and K9. After lyophilization, test vaccine 1 had avirus content of 10⁶.3 TCID₅₀ /ml, while the virus content of vaccine 2amounted to 10⁷.0 TCID₅₀ /ml.

In the recommendations of the WHO, "Laboratory Techniques in Rabies",3rd edition, Geneva, virus vaccines should have a minimum titer of 10⁵.2LD₅₀ or TCID₅₀. In the vaccines prepared according to the invention thevirus concentration was more than ten times higher than the requiredminimum concentration.

EXAMPLE 3

Adaptation of a production strain and preparation of an inactivatedvaccine for use in animal.

As starting material the rabies virus strain Flury LEP was used, whichcan be obtained from the American Type Culture Collection. The strainwas used in the form of a lyophilized, virus-containing chick-embryohomogenized product adapted to the cell strain Wi38. The adaptation toprimary chick-embryo fibroblast cells was performed according to theinvention by using for the series passages dilutions of the infectionmaterial slowly progressing in powers of ten in dependence on thepercentage of infected cells. The seed material for the preparation ofthe inactivated commercially available vaccine Madivak was prepared frompassage C22 (dilution passage 10⁻³.0). It had a virus titer of 10⁷.2.According to the invention, the vaccine was prepared with a 10⁻³.0dilution of the seed material or with an intermediate passage treatedaccording to the invention from the seed material prepared as describedin Example 1.

The vaccine had an NIH value of 2.6, while a vaccine prepared inaccordance with the state of the art, which had been obtained from othertissue cultures (cell strain PK 15) was found to have an antigen valueof 0.4 in the NIH test.

What is claimed is:
 1. In a process for preparing a rabies vaccine, the improvement wherein the rabies virus is first adapted to chick-embryo fibroblast cell cultures using a suspension containing the rabies virus in progressing dilutions from 10⁻¹ to 10⁻⁵ for the inoculation of the cell culture passages and then inoculating growth cultures with a suspension containing the adapted rabies virus in a dilution from 10⁻² to 10⁻⁵ for multiplication of the virus.
 2. A process as claimed in claim 1, wherein during the passages the temperature is maintained in the range of from 30° to 38° C.
 3. A process as claimed in claim 1, wherein during the passages the temperature is maintained in the range of from 32° to 37° C.
 4. A process as claimed in claim 1, wherein chick-embryo fibroblast cells of SPF eggs are used for the vaccine production. 